This invention pertains generally to liquid chromatography and more particularly to apparatus and methods for pretreating the solvents employed.
Liquid chromatographic systems generally employ a carrier liquid solvent or solvent mixture which is delivered, often at high pressure, to a column. The column may be of the type that is packed with stationary phase particles, or may be an open tube whose inside walls are coated with stationary phase material (i.e., so called "capillary columns"). The material to be analyzed is injected into the flow of the liquid carrier as it passes to the column. The solvents employed may be non-polar, polar or of intermediate polar properties. The particular solvent or solvents used to prepare the carrier are selected according to the characteristics of the material to be analyzed, the nature of the column, and the overall objectives of the analysis. In instances where the presence of dissolved gases, such as air, is objectionable, it has been the practice to effect removal of substantially all of the gases from the solvent by heating to boiling temperature, by subjecting the solvent to a partial vacuum, by ultrasonics, or combinations of such treatments. The simpler chromatographic systems employ a pump of the positive displacement type, which delivers the carrier to the column at pressures of a few p.s.i. to 7000 p.s.i. or higher, depending on the type of column used and the flow rate employed. A typical pressure range is from 500 to 5000 p.s.i. Such systems may employ a carrier comprising a single solvent, or a binary or tertiary solvent mixture in an isocratic mode (the solvent composition constant) or a gradient mode (the solvent composition programmed with time). The latter systems may employ two or more pumps delivering separate solvents to a mixer, with the solvent mixture (i.e. carrier) then being delivered to the column. Another gradient system makes use of relatively low pressure metering pumps or proportioning valves which deliver solutions in the proportions desired to a manifold and/or mixer, and from thence to the inlet of a single high pressure positive displacement pump which delivers the solvent mixture to the column. In both instances the presence of bubbles in the solvent or solvent mixtures may cause difficulties such as errors in the flow rates from the pumping means employed and errors in the composition of the carrier. Both flow and composition errors degrade chromatographic performance in terms of retention time precision, peak area precision and detector noise level. Particularly with respect to gradient systems of the type which employ a single high pressure positive displacement pump which delivers the carrier at high pressure directly to the column, bubbles tend to form on the way into or in the high pressure pump, due to gases such as air present in one or more of the solvents supplied to the manifold or mixing chamber, which is at relatively low pressure. Such bubbles cause excessive compliance in the high pressure pump chamber, thus causing increased flow rate errors and pulsation. Prior methods of treating the solvents to eliminate dissolved air are subject to certain disadvantages when applied to gradient systems. The hardware required for such pretreatment is relatively elaborate and cumbersome in operation and does not maintain a steady state degassed condition. For example, after a partial vacuum is applied and then released (it is not desirable to maintain a vacuum on the solvent during pumping), the gas concentration of various gases in the solvent, usually the air gases, starts to increase and in time reaches saturation level. This increase in gas concentration not only increases the probability of bubble formation, but in addition causes other undesirable results. Among these are the following: The presence of oxygen in the solvent can cause oxidation and degradation of certain samples, columns, plumbing connections and solvents. For example, tetrahydrofuron solvent forms peroxides in the presence of oxygen, and these cause absorbance of ultraviolet (UV) light, resulting in drifting baselines when UV photometric detectors are used. Oxygen itself can cause detecting drift due to its own UV absorbancy, and to its ability to quench fluorescence (in the case of fluorescent detectors). Quenching also results in nonreproducible detector response, and therefore to poor quantitative accuracy in chromatographic analysis. Oxygen can also cause a high background current, drift and noise in certain detectors, such as electrochemical detectors and electron capture detectors. The presence of carbon dioxide in the solvent can cause the pH value (acidity) of the solvent to change. This in turn affects the retention time of some eluted peaks and changes their UV absorption characteristics. The result is poor quantitative analytical precision. Also, the prior methods do not permit uninterrupted chromatography, and may not maintain steady state gas concentrations.